Dinucleoside Tetraphosphates—Pharmacology
Bis-adenosine tetraphosphate (Ap4A, P1,P4-di(5′,5″-adenosine)tetraphosphate, see structure 1) is the most important member of the class of bis-nucleoside polyphosphates. These nucleotides are ubiquitously found in variety of cells, and intracellular compartments, and appear to play important roles as both intra- and extracellular messengers in a variety of signaling events. In particular, they have been implicated in cellular stress response, blood pressure regulation, and insulin and glucose level regulation. Bis-adenosine polyphosphates, and in particular Ap4A, are involved in active inhibition of platelet aggregation by high affinity binding to platelet P2T receptors (Chan, et al., Proc. Natl. Acad. Sci. USA 94, 4034-4039, 1997). Ap4A and its phosphonates, e.g. “Ap2CHClp2A”, and the thio-analog, e.g. “Ap(S)pCHClpp(S)A” (see structure 2), have been proposed as possible agents for treatment of arterial thrombosis (Chan et al., op cit.).

Synthetic dinucleoside tetraphosphates have been studied for other indications, e.g., Up4U for chronic obstructive pulmonary disease (U.S. Pat. No. 5,635,160) and for prevention and treatment of pneumonia in immobilized patients (U.S. Pat. No. 5,763,447).
Dinucleoside Tetraphosphates—Synthesis
The first synthesis of Ap4A, along with other symmetrical dinucleoside polyphosphates, was reported by the group of Moffatt (J. Org. Chem. 30, 3381-3385, 1965) in their classic work on the dismutation of nucleoside polyphosphates. By reaction of 2 equivalents of AMP, activated as the phosphoromorpholidate, with one equivalent of pyrophosphate, Moffatt obtained Ap2A (8%), Ap3A (18%), Ap4A (23%), Ap5A (4%), along with ATP (7%), adenosine 5′-tetraphosphate (Ap4, 8%), and minor amounts of AMP and ADP.
Reaction of an excess of an activated nucleoside monophosphate with pyrophosphate remains the main synthetic approach to the synthesis of dinucleoside tetraphosphates and their dithio analogs:

For example Tarussova (Nucl. Acids Res. Symp. Ser. 14, 287-288, 1984) used carbonyldiimidazole (CDI) to activate AMP to the imidazolide. Blackburn used diphenylphosphoryl chloride as activating agent for thio-AMP to synthesize, inter alia, Ap(S)p2p(S)A in 24% yield (Tetrahedron Lett. 31, 4371-4374, 1990). In another approach, the AMP, activated as the morpholidate, was reacted with ATP (Ap4A and Other Dinucleoside Polyphosphates; McLennan, A. G. Ed. CRC Press: Boca Raton, Fla., pp 305-342, 1992). Direct catalyzed coupling of nucleoside diphosphates, and reaction of nucleoside triphosphates with nucleoside monophosphates have been reported (U.S. Pat. No. 6,765,090), but in only 10-15% yields.
All of these methods result in formation of considerable amounts of byproducts, such as nucleoside mono, di-, tri-, tetra- and even higher polyphosphates, together with the corresponding dinucleoside di-, tri-, and pentaphosphates. This makes the isolation and purification of the product difficult and time consuming. The chromatographic techniques utilized for that purpose are rarely suitable for large scale preparations.
Dinucleoside Tetraphosphonates—Synthesis
Reaction of an excess of activated nucleoside monophosphate with bisphosphonates remains the main synthetic approach to the synthesis of dinucleoside tetraphosphonates:

The group of Tarussova (Nucl. Acids Res. Symp. Ser. 14, 287-288, 1984) used carbonyldiimidazole (CDI) to activate AMP to the imidazolide, and condensed the imidazolide with methylenediphosphonate to give AppCH2ppA in 30% yield. The group of Blackburn used the Moffatt morpholidate method to prepare the same compound, along with other halomethylene analogs, in yields from 32 to 36% (Blackburn et al., In: Ap4A and Other Dinucleoside Polyphosphates; McLennan, A. G. Ed. CRC Press: Boca Raton, Fla., pp 305-342, 1992). They also used diphenylphosphoryl chloride as activating agent for thio-AMP to synthesize Ap(S)pCH2 pp(S)A, Ap(S)pCF2pp(S)A, and Ap(S)pCHFp(S)A in yields from 24 to 54% (Tetrahedron Lett. 31, 4371-4374, 1990).
Another possible approach is to react activated AMP with ATP analogs. The group of Blackburn used this method to prepare AppCCl2ppA from AppCH2p and AMP morpholidate in 46% yield (Ap4A and Other Dinucleoside Polyphosphates, loc cit).
All of these methods result in formation of considerable amounts of byproducts, such as nucleoside mono, di-, tri-, tetra- and even higher polyphosphonates, together with the corresponding dinucleoside di-, tri-, and pentaphosphonates. This makes the isolation and purification of the product difficult and time consuming. The chromatographic techniques utilized for that purpose are rarely suitable for large scale preparations.
The low yields and high cost of preparing potential therapeutic analogs of dinucleoside tetraphosphates and tetraphosphonates and related compounds have hampered further testing and development of this class.